Expression of membrane-bound carbonic anhydrase isozyme XII in mouse and rat tissues
Master’s thesis
Institute of Medical Technology
University of Tampere
May 2006
Piia Halmi
Acknowledgements
This study was carried out at the Institute of Medical Technology, University of Tampere. I want to thank Professor Seppo Parkkila and Docent Jari Honkaniemi for their excellent guidance and patience. I also want to thank Leena Honkaniemi, Jonna Hämäläinen and Aulikki Lehmus for their excellent and also crucial technical assistance.
Granada, May 2006
Piia Halmi
PRO GRADU – TUTKIELMA
Paikka: TAMPEREEN YLIOPISTO
Lääketieteellinen tiedekunta
Lääketieteellisen teknologian instituutti
Hiilihappoanhydraasien ja hemokromatoosin tutkimusryhmä Tekijä: HALMI, PIIA MARIKA
Otsikko: Solukalvoon kiinnittyvän isoentsyymi XII ilmentyminen hiiren ja rotan kudoksissa
Sivumäärä: 93 s.
Ohjaajat: Professori Seppo Parkkila ja Dosentti Jari Honkaniemi Tarkastajat: Professori Markku Kulomaa ja Professori Seppo Parkkila Aika: Toukokuu 2006
Tiivistelmä
Tutkimuksen tausta ja tavoitteet: Hiilihappoanhydraasi XII (CA XII) on solukalvoon kiinnittyvä isoentsyymi, joka ilmentyy joissakin ihmisen normaaleissa soluissa sekä yliekspressoituu joissakin syöpäsoluissa. CA12 geenin on todettu yliekspressoituvan joissakin syöpäkudoksissa hypoksian johdosta. Tässä tutkimuksessa selvitettiin CA XII:n ilmetymistä hiiren ja rotan elimistössä. Tutkimuksen tavoitteena oli tutkia ensin CA XII esiintymistä hiiren kudoksissa, ja myöhemmin keskittyä ilmentymisen tarkempaan tutkimiseen aivokudoksissa tunnetulla rottamallilla.
Tutkimusmenetelmät: Lähetti-RNA:n transkriptiota tutkittiin hiiren kudoksissa PCR:n avulla, ja rotan kudoksissa käänteiskopiointi-PCR:n (RT-PCR) avulla. Hiiren CA XII – proteiinia tutkittiin immunohistokemialla (biotiini-strepavidiini-menetelmä). Rotan aivokudoksia tutkittiin kainaattihapon aiheuttaman epilepsikohtauksen jälkeen sekä in situ -hybridisaation että northern blot -menetelmän avulla. In situ -hybridisaatiolla paikannettiin ilmentyminen, ja northern blot -menetelmällä kvantitoitiin ilmentymistä.
Tutkimustulokset: CA XII mRNA ilmentyy mm. hiiren munuaisissa, aivoissa, keuhkoissa, kivespusseissa ja sikiöissä. CA XII -proteiinilla on erittäin rajoittunut ilmentymiskenttä (munuainen, paksusuoli). Rotan aivoissa CA XII mRNA ilmentyy lähinnä choroid plexus:ssa ja aivokuoressa. Kainaattihappo stimuloi CA XII ilmentymistä kortikaalipinnalla.
Johtopäätökset: Runsas CA XII ekspressio hiiren munuaisissa ja paksusuolessa luultavasti selittää proteiinin merkitystä sekä kehon ioni- että pH-tasapainossa.
Kainaattihapon aiheuttaman stimuloinnin fysiologinen merkitys jäi epäselväksi, mutta CA XII:n runsas ilmentyminen choroid plexus:en alueella kertoo luultavasti nalogisesta toiminnasta CA II:n kanssa. CA II osallistuu aivoselkäydinnesteen eritykseen.
MASTER´S THESIS
Place: UNIVERSITY OF TAMPERE
Faculty of Medicine
Institute of Medical Technology
Carbonic anhydrase and hemochromatosis research group Author: HALMI, PIIA MARIKA
Title: Expression of membrane-bound carbonic anhydrase isozyme XII in mouse and rat tissues
Pages: 93 pp.
Supervisors: Professor Seppo Parkkila and Docent Jari Honkaniemi Reviewers: Professor Markku Kulomaa and Professor Seppo Parkkila Date: May 2006
Abstract
Background and aims: Carbonic anhydrase XII (CA XII) is a membrane-bound isozyme expressed in some normal human tissues, upregulated in some cancers, and is showed to be a hypoxia-inducible gene product. The expression of CA XII mRNA has been demonstrated in mouse kidney. This study concentrated on the research of the expression of CA XII in all mouse tissues, and later on in rat brain tissues.
Methods: mRNA transcription was studied by PCR in mouse tissues and by reverse transcriptase-PCR (RT-PCR) in rat tissues. CA XII protein in mouse tissues was studied by Immunohistochemistry (biotin streptavidin complex method). The studies on rat brain tissues were made by in situ hybridization and northern blot methods after kainic acid- induced status epilepticus. In situ hybridization showed the location of the expression where as northern blot served as a measure of quantity of the expression.
Results: CA XII mRNA is expressed in mouse kidney, brain, lung, testis and embryos. In embryos the expression became stronger with increasing age indicating developmental regulation. CA XII protein has a very limited expression distribution (mouse kidney and colon). In rat brain tissues CA XII mRNA is expressed mainly in dentate ganule cells, cortex and choroid plexus. Kainic acid stimulated the expression throughout the cortical layer.
Conclusions: The high expression of CA XII in mouse kidney and colon suggests a role for CA XII in the maintenance of body ion and pH homeostasis in the mouse. Kainic acid stimulates CA XII expression throughout the cortical layer. The physiological significance of the observed cortical induction of CA XII remains obscure, but the high expression of CA XII in the choroid plexus suggests an analogous role for this membrane-bound isozyme. CA II is known to participate in CSF secretion.
CONTENTS
ABBREVIATIONS ...7
1. INTRODUCTION ...9
2. REVIEW OF THE LITERATURE ...11
2.1. ENZYMES...11
2.2. CARBONIC ANHYDRASES...11
2.2.1. α-CARBONIC ANHYDRASES...15
2.2.1.1. Cytoplasmic carbonic anhydrases ...15
2.2.1.2. Mitochondrial and secretory carbonic anhydrases ...19
2.2.1.3. Membrane-associated carbonic anhydrases ...21
2.3. CARBONIC ANHYDRASE XII...23
2.3.1. General aspects...23
2.3.2. Expression of CA XII in normal tissues ...25
2.3.3. CA XII in tumors ...27
2.3.4. Von Hippel-Lindau...28
2.4. ACATALYTIC CARBONIC ANHYDRASES...30
2.5. CARBONIC ANHYDRASES IN NERVOUS SYSTEM...31
2.5.1. Formation of cerebro-spinal fluid (CSF) ...34
2.6. CARBONIC ANHYDRASE INHIBITORS...37
2.6.1. CA inhibitors in the nervous system...39
2.7. KAINIC ACID INDUCED STATUS EPILEPTICUS...41
2.7.1. Status epilepticus ...41
2.7.2. Kainic acid induced status epilepticus...44
3. AIMS OF THE RESEARCH ...46
4. METHODS ...47
4.1. EXPRESSION OF CA XII IN MOUSE TISSUES...47
4.1.1. Immunohistochemistry ...47
4.1.1.1. Animal treatments and tissue preparations ...47
4.1.1.2. Production of polyclonal rabbit antibody ...47
4.1.1.3. Immunohistochemical method ...47
4.1.2. PCR method...49
4.1.2.1. cDNA preparations ...49
4.1.2.2. PCR method...50
4.2. EXPRESSION OF CA XII IN RAT TISSUES...52
4.2.1. RT-PCR ...52
4.2.2. In situ hybridization and northern blotting ...52
4.2.2.1. Animal treatments and tissues preparations ...52
4.2.2.2. Extraction of mRNA ...53
4.2.2.3. Oligonucleotide probe preparation ...54
4.2.2.4. In situ hybridization method ...54
4.2.3. Northern Blotting...55
5. RESULTS ...56
5.1. EXPRESSION OF CA12 GENE IN MOUSE AND RAT TISSUES...56
5.2. DISTRIBUTION OF CA XII PROTEIN IN MOUSE TISSUES...57
5.3. IN SITU HYBRIDIZATION AND NORTHERN BLOTTING...62
6. DISCUSSION ...64
6.1. EXPRESSION OF CA XII IN MOUSE TISSUES...64
6.2. EXPRESSION OF CA XII IN RAT BRAIN...66
7. CONCLUSIONS...70
8. REFERENCES ...72
ABBREVIATIONS
AE anionic exchanger
AQP aquaporin
ARNT aryl hydrocarbon receptor nuclear translocator
BBB bloob-brain barrier
BSA bovine serum albumin
CA carbonic anhydrase
CA12 carbonic anhydrase 12 (refers particularly to the human gene) Car7 carbonic anhydrase 7 (refers particularly to the mouse gene) CA-RP carbonic anhydrase related protein
CD collecting duct
cDNA complementary deoxyribonucleic acid CNS central nervous system
CSF cerebrospinal fluid
Da Dalton
DAB 3,3’diaminobenzidine tetrahydrochloride
DEPC diethylpyrocarbonate
DNase deoxyribonuclease
DNA deoxyribonucleic acid
EDTA ethylenediaminetetraacetic acid
EEG electroencephalogram
G3PDH glyceraldehyde 3-phosphate dehydrogenase GABA γ-aminobutyric acid
GL glomerulus
Gly glysine
GPI glycosyl phosphatidylinositol HIF hypoxia inducible factor
His histidine residue
IEG immediate-early gene
mRNA messenger ribonucleic acid
NMDA N-methyl-D-aspartate PBS phosphate-buffered saline PCR polymerase chain reaction PCT proximal convoluted tubule pVHL von Hippel-Lindau protein
RNA ribonucleic acid
RNAse ribonuclease
RPTP receptor protein tyrosine phosphatase
RT-PCR reverse transcriptase polymerase chain reaction SDS sodium dodecyl sulphate
Ser serine
Ub ubiquitin
VBC VHL/Elongin B/Elongin C
VEGF vascular endothelial cell growth factor
VHL von Hippel-Lindau
1. INTRODUCTION
Carbonic anhydrases (CAs) are a family of zinc metal enzymes which catalyze the reversible spontaneous hydration of carbon dioxide (CO2) (Sly & Hu, 1995). There are at least 13 known α-CAs which are characterized in animal kingdom and three CA-related proteins which lack enzymatic activity. A recent study shows a novel member of this gene family, carbonic anhydrase XV, which seems to exist as a non-processed pseudogene in humans and chimpanzees (Hilvo et al., 2005).
Carbonic anhydrase XII, CA XII, is a 354-amino acid polypeptide type I transmembrane protein with its active extracellular domain containing three zinc-binding histidine residues which can be found in active CAs and two potential sites for asparagine glycosylation. CA XII protein sequence has a sequence identity of 30-42 % to other CAs (Türeci et al., 1998) and it exists as a dimer in both solution and the crystal. The CA12 gene has been identified as a von Hippel-Lindau target gene, suggesting a potential role for CA XII in von Hippel-Lindau carcinogenesis (Ivanov et al., 1998). Recently, CA XII has been found to be a hypoxia-inducible protein (Ivanov et al., 2001; Wykoff et al., 2001; Watson et al., 2003), possibly explaining its upregulation in certain tumors. These recent reports suggest that CA XII may be an excellent marker for hypoxia in tumors.
CA XII has a wide distribution spectrum in normal tissues (Ivanov et al., 1998;
Karhumaa et al., 2000; Kivelä et al., 2000; Parkkila et al., 2000; Türeci et al., 1998). CA XII mRNA is expressed in numerous human tissues such as aorta, bladder, brain, colon, esophagus, kidney, lung, mammary gland, ovary, prostate, pancreas, rectum, testis, trachea and uterus (Türeci et al., 1998; Ivanov et al., 1998; Kivelä et al., 2000). In the brain CA12 gene is expressed only in the corpus striatum (caudate nucleus and putamen) (Ivanov et al., 1998; 2001). Immunohistochemical studies show CA XII expression in the human reproductive tissues, colon, mesothelial cells and the coelomic epithelium of the body cavity, and kidney, sweat glands of the skin, epithelium of the breast, salivary glands, upper respiratory system, nose, and pancreas. Limited expression can be found also in the prostate, vas deferens, and transitional mucosa of the renal pelvis. In the
gastrointestinal tract CA XII expression is limited to the surface epithelium of the large intestine. In the nervous system CA XII immunoreactivity is found in the choroid plexus, in limited numbers of ganglion cells of the cortex, in the posterior lobe of the pituitary glands, and in the remnant of Rathke’s pouch (Ivanov et al., 2001; Karhumaa et al., 2000;
2001; Kivelä et al., 2000; Parkkila et al., 2000).
The expression of CA12 in multiple cancers has been shown to be in high level (Ivanov et al., 2001) but its function in normal and malignant tissues is not yet exactly known. The CA XII overexpression has been shown in some human renal cancer cells (Türeci et al., 1998). Based on its expression pattern CA XII could serve as a biomarker for some malignant tumors (non-small cell lung carcinoma) and could be considered a potential target for novel therapeutic applications (Ivanov et al., 1998).
This study is focused on the expression and distribution of CA XII in mouse tissues and rat brain tissues. We also evaluate CA XII expression in the rat brain after kainic acid induced epileptic seizures.
2. REVIEW OF THE LITERATURE 2.1. ENZYMES
Enzymes are special cases among other proteins in that they bind and chemically transform (catalyze reactions) other molecules. Their catalytic power is often far greater than that of synthetic or inorganic catalysts. The molecules acted upon by enzymes are called reaction substrates, and the ligand-binding site is called the active site or catalytic site. Enzymes have a high degree of specificity for their substrates. They accelerate chemical reactions, and function in aqueous solutions under very mild conditions of temperature and pH. The study of enzymes is of great importance because a deficiency or total absence of one or more enzymes or their excessive activity may cause various diseases (Nelson & Cox, 2000).
Some enzymes do not require any chemical groups for activity other than their amino acid residues, while others require an additional chemical component called a cofactor (one or more inorganic ions, e.g. Fe2+, Mn2+, or Zn2+) or a complex organic or metalloorganic molecule called a coenzyme. There are even some enzymes which require both a coenzyme and one or more metal ions for activity. In the case of carbonic anhydrases, the enzymes require Zn2+ ion in their catalytic site. Catalytically active enzyme is called a holoenzyme, and its protein part apoenzyme or apoprotein. A coenzyme or a cofactor bound tightly or covalently to the enzyme protein is called a prosthetic group. Enzyme activity can be altered also by modifying the enzyme covalently by phosphorylation or glycosylation (Nelson & Cox, 2000).
2.2. CARBONIC ANHYDRASES
The carbonic anhydrases catalyze the reversible spontaneous hydration of carbon dioxide (CO2) with high efficiency, a reaction which underlies many diverse physiological processes in animals, plants, archaebacteria, and eubacteria such as photosynthesis, respiration, renal tubular acidification, and bone resorption. The carbon dioxide produced
by oxidation of organic fuels in mitochondria is hydrated to form bicarbonate (HCO3-) by carbonic anhydrase (figure 2.1.).
− +
−
− +
+
⇔
+
⇔
⇔ +
2 3 3
3 3
2 2
2
CO H
HCO
HCO H
CO H O H CO
Figure 2.1. Hydration of carbon dioxide (CO2) to bicarbonate (HCO3-).
Carbon dioxide is not very soluble in aqueous solution, and it would form bubbles of CO2
in the tissues and blood if it were not converted to bicarbonate. The hydration of CO2 to bicarbonate results in an increase in the H+ concentration in the tissues and therefore a decrease in pH. Bicarbonate in the erythrocytes reenters the blood plasma for transport to the lungs (figure 2.2.).
Figure 2.2. The function of chloride-bicarbonate exchanger and carbonic anhydrase in the erythrocyte membrane (Modified from Nelson & Cox, 2000).
Compared to CO2, HCO3- is much more soluble in blood plasma. Therefore, the generation of HCO-3 ions increases the capacity of the blood to carry CO2 from the tissues to the lungs. In the lungs HCO3- reenters the erythrocyte and is converted to CO2,
is influenced by pH and CO2 concentration, so the interconversion of CO2 and bicarbonate is of great importance to the regulation of oxygen binding and release in the blood. About 20% of the total CO2 and H+ formed in the tissues is transported by haemoglobin to the lungs and kidneys. The binding of these ions is inversely related to oxygen binding. At the low pH and high CO2 concentration (peripheral tissues) the affinity of haemoglobin for oxygen decreases and oxygen is released. In the capillaries (lung tissues) CO2 excretion causes the raise of pH, and the affinity of haemoglobin for oxygen increases. More oxygen is transported to the peripheral tissues (Nelson & Cox, 2000).
Figure 2.3. Oxidation of carbon dioxide (CO2) to form bicarbonate (HCO3-) with the help of a zinc ion. Zinc is bound to three histidines in carbonic anhydrase. Water is ionised to a hydroxide ion and this is stabilised by the Zn2+ ion. CO2 then enters the active site and is attacked by OH−, forming a carbonate ion, which is then released, regenerating the enzyme.
All enzymatically active carbonic anhydrases contain a zinc ion (Zn2+), which is critical for the catalytic activity (figure 2.3.). The zinc ion is coordinated by three histidine residues (Stams & Christianson, 2000). The transfer of H+ from the the zinc-bound water molecule to the solution generates OH-. The central catalytic step is a reaction between
this zinc-bound OH- ion and CO2 leading to a HCO3- ion which the water molecule displaces from the metal ion (Lindskog & Silverman, 2000; Supuran, 2004).
Carbonic anhydrases are clustered into three distinct gene classes (α, β, and γ) which have evolved independently and have no sequence homology (Hewett-Emmet & Tashian, 1996). All the 13 characterized CAs of the animal kingdom belong to a single gene family of the α-carbonic anhydrases (α-CAs). These isozymes have been found in all mammalian tissues and cell types and they show characteristic cellular localization; they are found in cytosol (CA I, CA II, CA III, CA VII and CA XIII) (Sly and Hu, 1995;
Earnhardt et al., 1998; Lehtonen et al., 2004), mitochondria (CA VA and CA VB) (Fujikawa-Adachi et al., 1999), secretory granules (CA VI) (Murakami & Sly, 1987), and plasma membrane (CA IV, CA IX, CA XII, CA XIV and CA XV) (Zhu and Sly, 1990;
Pastorek et al., 1994; Türeci et al., 1998; Ivanov et al., 1998; Mori et al., 1999; Hilvo et al., 2005) (table 2.1.).
Table 2.1. Carbonic anhydrases and their subcellular localization.
Carbonic anhydrase
Subcellular localization I cytoplasmic II cytoplasmic III cytoplasmic IV
membrane- bound VA mitochondrial VB mitochondrial
VI secreted VII cytoplasmic
IX transmembrane XII transmembrane XIII cytoplasmic XIV transmembrane
XV
membrane- bound
There are also other known inactive isoforms which have homologous domains to active carbonic anhydrases (CA-RP VIII, CA-RP X, CA-RP XI, RPTP- β and RPTP- γ) which do not appear to have any activity similar to earlier ones. This is due to changes in the active site histidine (His) residues which are critical for carbon dioxide hydration catalysis (zinc ion is not bound when His residues are missing). These isoforms are widespread in mammalian body tissues (Nishimori, 2004; Taniuchi et al., 2002a; 2002b).
The α-CA isozymes differ in their tissue distribution, subcellular localization, and kinetic properties. Some isozymes such as CA II are expressed in a number of different tissues, whereas others (e.g. CA VI, IX and XIV) show a more limited distribution. All active isozymes are expressed in the alimentary tract, although the cellular localization is unique for each isozyme (Parkkila et al., 1994b; Fleming et al., 1995; Sly and Hu, 1995; Parkkila
& Parkkila, 1996; Pastoreková et al., 1997; Kivelä et al., 2000).
Distinct α-CAs can be found in protostomes (Drosophila melanogaster, Caenorhabditis elegans, Caenorhabditis briggsae, Galeocerdo cuvieri). Several plant chloroplast CAs and certain eubacteria (Escherichia coli and Synechococcus) CAs have been shown to belong to a distinct gene family, β-carbonic anhydrases (β-CAs) (Fukuzawa et al., 1992;
Guilloton et al., 1992; 1993). The third distinct gene family is represented by γ-CAs which includes at least the isoforms of archaebacterium Methanosarcina thermophila (Alber & Ferry, 1994). This division of CA isoforms into three separate classes is only a simple assumption because the plant Arabidopsis for example has homologues of all of these three enzyme families (Hewett-Emmett & Tashian, 1996).
2.2.1. α-CARBONIC ANHYDRASES 2.2.1.1. Cytoplasmic carbonic anhydrases
The carbonic anhydrases I and II are both quite widely expressed cytoplasmic isozymes. The CA1, CA2 and CA3 genes exist in a cluster on the chromosome 8q22. CA I is expressed in the α-cells of the endocrine Langerhans’ islets (Parkkila et al., 1994b). It
Da. CA I is expressed at low levels in the A cells of Langerhans islets, the epithelium of the large intestine, corneal epithelium, the lens of the eye, the placenta and foetal membranes (Muhlhauser et al., 1994; Parkkila et al., 1994; Sly & Hu, 1995), and overexpressed in chronic myeloproliferative disorders (Bonapace et al., 2004a). The CA I and II are expressed in human colonic mucosa in the non-goblet columnar cells lining the main lumen in the upper half of the crypts (Bekku et al., 1998; Davenport & Fisher 1938;
Davenport, 1939; Lönnerholm et al., 1985; O’Brien et al., 1977; Sato et al., 1980;
Parkkila et al., 1994b). The expression has been demonstrated to increase gradually over time during differentiation of the mucosal cells, and therefore they could function as useful markers for the differentiation of enterocytes in the colonic mucosa. CA I is five times as abundant as CA II in human erythrocytes. Because CA I is a low activity isozyme, it contributes only about 50% of the total activity in these cells, which could explain why CA II deficient people do not have defects in erythrocytes (Dodgson et al., 1988).
Figure 2.4. Function of carbonic anhydrase II in the proximal tubule cell in the kidney
Carbonic anhydrase II is a best known isoenzyme and is located almost in all organs. It has a molecular weight of 30,000 Da and is highly expressed in the intercalated cells of the late distal tubule, the collecting tubule, and the collecting duct of the kidney and some expression has been reported in the loop of Henle, the proximal tubules (figure 2.4.), and the principal cells of the collecting ducts (Brown et al., 1983; Brown & Kumpulainen, 1985; Holthöfer et al., 1987; Lönnerholm et al., 1986; Lönnerholm & Wistrand, 1984;
Parkkila et al., 1994b; Sato & Spicer, 1982; Spicer et al., 1982; Spicer et al., 1990). Sly et al. (1983) reported renal tubular acidosis in patients with CA II-deficiency syndrome. CA II-deficient mice produced by chemical mutagenesis also had impaired renal acidification (Lewis et al., 1988). More than 95 per cent of renal CA activity is cytosolic and corresponds to CA II, while 3-5 per cent is membrane associated (McKinley & Whitney, 1976; Wistrand & Kinne, 1977). In the hepatic bile ducts, CA II facilitates the alkalization of the bile (Parkkila & Parkkila, 1996) and is involved in bile acidification occurring in the gallbladder (Juvonen et al., 1994). CA II is present in the human epididymis and ductus deferens where it could be involved in the acidification of the epididymal fluid. It seems to be abundant in the brush border regions of the surface epithelium. The bicarbonate of the ejaculate is produced by CA II in the epithelium of ductus deferens and seminal vesicle. The bicarbonate ions present in the seminal fluid may induce sperm motility in the vagina and cervix and also buffer the low pH of the vaginal milieu. The pH of the CSF produced in the choroid plexus is regulated by CA II.
CA II catalyzes also the hydration of CO2 to HCO3- in erythrocytes and the HCO3-
production to saliva (Chedwiggen & Carter, 2000).
In 1974 Garg detected for the first time CA III in rat liver. This very low activity isozyme, CA III, is expressed also in adipocytes and skeletal muscle (Jeffery et al., 1980;
Kim et al., 2004a). It is one of the first isozymes detected by specific antibodies (Spicer et al., 1979; 1982; 1990). It functions in an oxidizing environment and it is the most oxidatively modified protein in the liver known so far (Cabiscol & Levine, 1995). CA III may provide protection from oxidative damage and may serve as a useful marker protein to investigate in vivo the mechanisms contributing to oxidative damage in the liver (Parkkila et al., 1999). Low levels of free radicals in cells over-expressing CA III may
affect growth-signalling pathways (Räisänen et al., 1999). The knock-out mouse model generated by Kim et al. (2004a) showed no morphological neither physiological abnormalities, and CA III deficient mice studied by Zimmerman et al. (2004) suggested a anti-oxidative role for this protein in the skeletal muscle.
The carbonic anhydrase VII is the most highly conserved α-CA. The sequence identity is ~95% between human and mouse homologues which suggests an important biological role for this enzyme (Sly & Hu, 1995). The CA7 gene has been localized to the chromosome 16q22-23 (Montgomery et al., 1991). The CA VII enzyme has been expressed in E. coli and shown to possess carbon dioxide hydrase activity (Lakkis et al., 1996). In situ hybridization has demonstrated CA VII expression in mouse brain at high level in the Purkinje cells, in the granular and molecular layers, at the pial surface and in the large neurons throughout the cortical layer, in the medial habenulae, in neurons of the thalamus, strongly expressed in the hippocampal formation, specifically in the pyramidal cells of Ammon’s horn and in the granular cells of the dentate gyrus, and in the choroid plexus and cerebrospinal fluid-containing channels (Lakkis et al., 1997). This distribution of mRNA in the mouse brain could suggest a non-specific but generalized function for CA VII in the mouse brain. CA activity plays an important role in cerebrospinal fluid production and in regulation of its ionic constituents and pH. The studies showed a very strong expression in neurons, hence the CA VII could be important in maintaining the metabolic activity of the neurons by the elimination and transport of CO2 produced by glycolysis and it could effect on neuroexcitation and susceptibility to seizures in many distinct ways. CA VII can be important in maintaining different membrane transport processes by facilitation of CO2 transport and regulation of transmembrane fluxes (correct distribution of chloride ions). Some connection between CA activity in the dorsal root ganglia with the cytochrome oxidase activity has been commented. This could effect on the electrical activity of the neurons and their energy requirements. High concentrations of CA are probably necessary in the growth and maturation of neurons. A peripheral nerve injury reduces CA enzyme content or its activity in dorsal root ganglion cells (Lakkis et al., 1997). Ruusuvuori et al. (2004) suggest a function for CA VII in a developmental process enabling synchronous firing of CA1 pyramidal neurons.
A novel carbonic anhydrase XIII, CA XIII, has been recently characterized (Lehtonen et al., 2004). CA XII mRNA is expressed in the human thymus, small intestine, spleen, prostate, ovary, colon, and testis and in the mouse spleen, lung, kidney, heart, brain, skeletal muscle, and testis. Distribution of CA XIII shows similarities with that of other cytosolic isozymes. Computer modelling of CA XIII structure revealed that it is a globular molecule with high structural similarity to cytosolic isozymes, CA I, II, and III.
Furthermore, kinetic studies have demonstrated catalytic activity similar to mitochondrial CA V and cytosolic CA I.
2.2.1.2. Mitochondrial and secretory carbonic anhydrases
Dodgson (1991) described the expression of CA V for the first time in the rat liver and kidney, and in the mitochondria of the liver and skeletal muscle of guinea pigs. Later studies have revealed that mammalian tissues contain two different mitochondrial CA isoforms, CA VA and CAVB (Fujikawa-Adachi et al., 1999). CA VA is expressed mainly in the liver, whereas CA VB has a wide expression distribution in many tissues except in the liver (Shah et al., 2000). The expression of human CA V cDNA in COS-7 cells produced a 34,000 DA precursor and 30,000 Da mature enzymes (Nagao et al., 1993). CA V is the second isozyme described in the endocrine pancreas, where its expression is solely confined to the β-cells (Parkkila et al., 1998). It has been proposed that mitochondrial CA regulates insulin secretion by providing bicarbonate ions for the pyruvate-malate shuttle operating in these cells. CA V is expressed also in brain tissue and in the gastrointestinal tract (Saarnio et al., 1999; Sato et al., 2002).
The mammalian liver expresses high levels of mitochondrial CA V and it has been implicated in two metabolic processes in the mitochondria of hepatocytes: ureagenesis and gluconeogenesis. CA V would supply bicarbonate for the first urea cycle enzyme, carbamyl phosphate synthetase I in ureagenesis and for pyruvate carboxylase in gluconeogenesis (Dodgson, 1991). CA V could have an important role also in lipogenesis ()pyruvate carboxylation) (Lynch et al., 1995; Hazen et al., 1996). CA inhibitors have
been observed to retard both of these processes in the livers of guinea pigs and rats (Dodgson et al., 1983; Metcalfe et al., 1985; Dodgson, 1991).
The only known secretory carbonic anhydrase of the CA gene family is CA VI. It is secreted from the salivary glands (Fernley et al., 1979; Feldstein & Silverman 1984;
Murakami & Sly 1987; Kadoya et al., 1987). The amino acid sequence of sheep CA VI has been reported (Fernley et al., 1988), and Aldred et al. (1991) have determined the cDNA sequence of human CA VI. A probable stabilization of CA VI in the environment of the alimentary tract is caused by an intramolecular disulfide bond formed by two cysteine residues (Fernley et al. 1988; Aldred et al. 1991; Parkkila et al. 1997). CA domain of CA VI is highly homologous to four other CAs (CA IV, CA IX, CA XII and CA XIV) and they form together a group of extracellular CAs (Fujikawa-Adachi et al., 1999b; Mori et al., 1999).
The molecular weight of CA VI varies depending on a species between 39 and 46 kDa (Parkkila & Parkkila, 1996). CA VI is produced in the serous acinar cells of the parotid and submandibular glands (Kadoya et al., 1987; Parkkila et al., 1990). The secretion of CA VI is controlled by autonomic nervous system (Fernley, 1991), and it follows circadian period (Kivelä et al., 1997; Parkkila et al., 1993; Parkkila et al., 1995). CA VI protects teeth from caries by accelerating the neutralization of the protons produced by cariogenic bacteria (Kivelä et al., 1999) and it is also involved in the neutralization processes in the upper gastrointestinal tract (Parkkila et al., 1997) and pancreas (Fujikawa-Adachi et al., 1999a). CA VI has been suggested to be linked with taste function (Thatcher et al., 1998) and furthermore as a trophic factor for the taste bud stem cells (Henkin et al., 1999b). Karhumaa et al. (2001) has reported the presence of CA VI in milk, and Kimoto et al. (2004) in the mouse nasal gland. CA VI has been suggested to have a mucosa-protective role also in the respiratory tract (Leinonen et al., 2004).
2.2.1.3. Membrane-associated carbonic anhydrases
The first membrane-bound isozyme described was carbonic anhydrase IV (CA IV) (Whitney & Briggle, 1982) which is a glycosyl phospatidylinositol (GPI)-anchored protein expressed widely in various tissues. The CA4 gene is located on chromosome 17 (Okuyama et al., 1992; 1993), and the molecular weight of the human CA IV protein is 35,000 Da (Zhu & Sly, 1990). The physiological role of CA IV is to facilitate the reversible hydration of carbon dioxide (CO2) at the sites of a rapid flux of CO2 and HCO3- across membranes (Sly & Hu, 1995).
CA IV is expressed on the luminal surface of pulmonary endothelial cells in lungs catalyzing the dehydration of bicarbonate to carbon dioxide (Fleming et al., 1993; Zhu &
Sly 1990). On the brush border membrane of the proximal tubular cells and on the cells of thick ascending limbs of Henle, in kidney, CA IV facilitates bicarbonate reabsorption (Brown et al., 1990; Zhu & Sly 1990). CA IV on the apical surface of the epithelial cells in the colon and in distal small and large intestine participates in ion and fluid transport (Fleming et al., 1995). CA IV is localized also in human epididymis and ductus deferens of the male genitourinary tract (Ghandour et al., 1992), in the capillary endothelium of skeletal and heart muscle (Sender et al., 1994; Sender et al., 1998), and on the plasma face of endothelial cells of several capillary beds (Parkkila et al., 1996; Pastoreková et al., 1997; Schwartz, 2002;). Human CA IV has been also localized in the luminal plasma membrane of the gallbladder and bile duct epithelium (Parkkila et al., 1996), pancreas, salivary glands (Fujikawa-Adachi et al., 1999a), in the brain capillary endothelial cells (Ghandour et al., 1992), in erythrocyes (Wistrand et al., 1999) and choriocapillaris of the eye (Hageman et al., 1991).
Carbonic anhydrase IX (CA IX) is a 54,000/58,000 Da mass transmembrane glycoprotein with a signal peptide, proteoglycan-related sequence, transmembrane segment, complete CA domain in the middle of its large extracellular segment, and a short intracellular tail (Pastorekova & Zavada, 2004). CA IX was first found as a tumor- associated antigen, MN, in the normal gastric mucosa and human carcinomas
plasma membrane of epithelial cells and also in some cases in the nucleus. A human CA9 gene has been mapped to chromosome 17 (Ivanov et al., 1998). The CA IX enzyme is expressed in the gastric epithelium (Pastorek et al., 1994), and the CA IX-positive cell types in the gastric mucosa were first defined by Pastoreková et al. (1997). CA IX expression has been observed also in the biliary epithelial cells (Parkkila & Parkkila, 1996; Pastoreková et al., 1997), and the presence of CA IX in the colonic enterocytes has been reported by Saarnio et al. (1998). After the discovery of the CA domain it was named as CA IX. The isozyme IX has restricted distribution (neoplastic cells) which could suggest its role in cell proliferation (also non-malignant) and transformation. A recent study on CA IX-deficient mice produced by targeted mutagenesis revealed that the enzyme deficiency results in a marked hyperplasia of mucus-producing cells in the gastric mucosa (Ortova Gut et al., 2002). These results suggest that CA IX is functionally implicated in gastric morphogenesis via the control of cell proliferation and differentiation.
Carbonic anhydrase XIV (CA XIV) is a transmembrane protein (Mori et al., 1999) with an amino-terminal signal sequence, a CA domain with high homology with other extracellular CAs, a transmembrane domain, and a short intracellular C-terminal tail. The CA14 gene has been mapped to chromosome 1q21 (Fujikawa-Adachi et al., 1999c).
Whittington et al. (2004) have suggested that the activity of CA XIV migth be higher than that of CA II. CA14 mRNA expression has been demonstrated in the human heart, brain, liver, spinal cord and skeletal muscle and a weak expression has been detected in the small intestine, colon, kidney, and urinary bladder by RNA dot blot analysis (Fujikawa-Adachi et al., 1999c). In the mouse kidney CA14 mRNA is expressed also in the apical and basolateral plasma membranes of the S1 and S2 segments of the proximal tubules, in the outer border of the inner stripe of the outer medulla and in the initial portion of the thin descending limb of Henle (Kaunisto et al., 2002). Parkkila et al. (2001) observed in their studies a strong expression of CA XIV in the neuronal membranes and axons in different parts of the human and mouse brain. This could suggest the role of CA XIV in modulation of excitatory synaptic transmission in the brain. A recent study has demonstrated an abundant expression of CA XIV at the plasma membrane of murine
hepatocytes (Parkkila et al., 2002) where it might regulate the pH and ion homeostasis between the bile canaliculi and hepatic sinusoids.
Most recently Hilvo et al. (2005) have described a novel member of -CAs called CA XV. At least eight species have genomic sequences encoding CA XV in which all the amino acid residues critical for CA activity are present. Apparently CA XV has become a non-processed pseudogene in humans and chimpanzees and studies with RT-PCR
confirmed that humans do not express CA XV. In mice positive expression of CA XV mRNA can be seen in the kidney, brain, and testis. Based on a phylogenetic analysis mouse CA XV is closely related to CA IV, and CA XV also shares several structural properties with CA IV, i.e., it is a glycosylated, GPI-anchored membrane protein, and it binds CA inhibitor. Similar to mouse CA IV, the catalytic activity of CA XV is low. CA XV is probably the first member of the -CA gene family which is expressed in several species but not in humans and chimpanzees.
2.3. CARBONIC ANHYDRASE XII
2.3.1. General aspects
Human CA XII was originally cloned and characterized by two groups independently (Türeci et al., 1998; Ivanov et al., 1998), in both cases as a gene whose mRNA is greatly upregulated in renal cell carcinomas. The cDNA sequence predicted a 354-amino acid polypeptide with a molecular mass of 39,448 Da. The CA12 gene has been mapped to chromosome 15q22 by fluorecence in situ hybridization (Türeci et al., 1998). CA XII is a one-pass, type I transmembrane protein with a 29-amino acid signal sequence (predicted cleavage between Gly-1 and Ser-1), 261-amino acid CA domain (homology domain), an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a 29-amino acid C-terminal cytoplasmic tail which contains two potential phosphorylation sites. CA XII protein sequence has a sequence identity of 30-42 % to other CAs (Türeci et al., 1998) and the extracellular domain contains three zinc-binding histidine residues (His-94, His-96 and His-119) which are present in the active sites of
Figure 2.5. Amino acid sequence of human catalytic CAs. The aligned sequences correspond to numbering of amino acids in CA I (numbers above). The conserved residues are bold-faced, and arrows above His-94, His-96 and His-119 indicate Zn binding site residues. (Modified from Lehtonen et al., 2004)
catalytically active CAs (figure 2.5.). Histidine residue 64 is also conserved and it has been shown to contribute to the efficiency of high-activity CAs by serving as a proton shuttle between the zinc-bound water molecule and surrounding buffer molecules. The extracellular domain contains two potential sites for aspargine glycosylation and four cysteine residues (Türeci et al., 1998).
The crystal structure of a secretory form of human CA XII at 1.55-Å resolution has been described, and in its native state, CA XII appears as a dimer (Whittington et al., 2001).
The activity of CA XII is moderate and is approximately same as the activity of CA I (Ulmasov et al., 2000). The CA12 gene has been identified as a von Hippel-Lindau target gene, suggesting a potential role for CA XII in von Hippel-Lindau carcinogenesis (Ivanov et al., 1998). Recently, CA XII has been found to be a hypoxia-inducible gene (Ivanov et al., 2001; Wykoff et al., 2001; Watson et al., 2003), possibly explaining its upregulation in certain tumors. These recent reports suggest that CA XII may be an excellent marker for hypoxia in tumors.
2.3.2. Expression of CA XII in normal tissues
CA XII has a wide distribution pattern in normal tissues (Ivanov et al., 1998; Karhumaa et al., 2000; Kivelä et al., 2000; Parkkila et al., 2000; Türeci et al., 1998). Its mRNA is expressed in several normal human tissues such as aorta, bladder, brain, colon, esophagus, kidney, liver, lung, lymph node, mammary gland, ovary, prostate, pancreas, peripheral blood lymphocytes, rectum, stomach, skeletal muscle, skin, spleen, testis, trachea and uterus (Türeci et al., 1998; Ivanov et al., 1998; Kivelä et al., 2000). In the brain, CA12 gene was found to be expressed only in the corpus striatum (caudate nucleus and putamen) (Ivanov et al., 1998; 2001).
Immunohistochemical studies have shown that CA XII protein is expressed in the human reproductive tissues (surface and glandular epithelial cells of the human endometrium, occasional epithelial cells in the uterine cervix, syncytiotrophoblasts of the placenta, epithelium of the efferent ducts, apical mitochonria-rich cells of the epididymal duct),
colon (basolateral plasma membrane of the epithelial cells), mesothelial cells, and kidney (renal proximal tubule, distal convoluted tubules, intercalacted cells of the collecting duct), sweat glands of the skin, epithelium of the breast, salivary glands (ductal cells, mucous cells), upper respiratory system (submucosal glands), nose (epithelial cells of Schneider’s membrane), pancreas (acinar cells). Limited expression can be found also in the prostate, vas deferens, and transitional mucosa of the renal pelvis. In the gastrointestinal tract CA XII expression is limited to the surface epithelium of the large intestine. In the nervous system, CA XII immunoreactivity is found in the choroid plexus, in limited numbers of ganglion cells of the cortex, in the posterior lobe of the pituitary glands, and in the remnant of Rathke’s pouch. (Ivanov et al., 2001; Karhumaa et al., 2000a, 2001; Kivelä et al., 2000; Parkkila et al., 2000).
CA XII is present in almost all epithelial cells of the efferent ducts and localized to the basolateral plasma membrane. It has been localized to the same cells as AQP1 in the excurrent tubule system which could suggest a role in ion transport and fluid reabsorption. The fluid leaving testis is modified and concentrated in the excurrent ducts by water reabsorption coupled to active transport of sodium and chloride ions. CA XII probably participates in these transport processes. Sodium and chloride ions are transported through the basolateral membrane via a Cl-/HCO3- exchanger and co- transporter Na+/HCO3- (NBC) (Jensen et al., 1999a;1999b). NBC probably needs CA to eliminate CO32- gradients across the membrane, and CA might prevent the accumulation of HCO3- like in the renal proximal tubule (Müller_Berger et al., 1997). The apical mitochondria-rich cells of the epididymal duct, where the CA XII expression was also demonstrated, are involved in acidification of the epididymal fluid (Brown et al., 1992;
Karhumaa et al., 2001; Martínez-García et al., 1995).
The staining with an antibody against a secreted form of human CA XII showed positive expression in the basolateral membranes of cells of the thick ascending limb, proximal and distal tubules and in the principal cells of the collecting ducts of the human kidney.
The cellular distribution of CA XII has suggested an important role for this isozyme in
normal renal physiology (such as acidification of urine) and regulation of water homeostasis (Kyllönen et al., 2003; Nielsen et al., 1993; Parkkila et al., 2000).
2.3.3. CA XII in tumors
The expression of CA XII in multiple cancers has been shown to be in high level (Ivanov et al., 2001), even though its function in normal and malignant tissues is not yet exactly known. The CA XII overexpression has been shown in some human renal cancer cells.
The renal tumour tissue had higher CA XII transcript expression levels than in the surrounding normal kidney tissue in 10 % of the patients with renal cell carcinoma (Türeci et al., 1998). The isozyme XII detected in renal cell carcinomas was shown by sequencing to be identical to that of a normal kidney. CA XII could acidify the immediate extracellular milieu surrounding the cancer cells in malignant tumors which could create a microenvironment leading to tumor growth and spread. The active site of the enzyme is located on the cell exterior, and this way it could regulate the extracellular pH in close proximity to the epithelium. The CA XII could be functionally coupled to an unidentified bicarbonate transporter to move bicarbonate across the basolateral membrane into the epithelial cells and to another transporter on the apical surface to secrete the bicarbonate ions. CA XII could also participate in a ligand-binding domain which is involved in transformation of tumor cells in a process that could be enhanced by the CA activity at the membrane of highly proliferating cells. If the CA activity at the membrane played a role in transformation, there could be a possibility to develop isozyme-specific CA inhibitors that could diminish the transforming potential of the membrane CA (McKiernan et al., 1997, Türeci et al., 1998).
The expression of CA XII is restricted to certain cell type and degree of differentiation within a given organ. CA XII shows membrane-associated expression in most oncocytomas and clear-cell carcinomas. CA XII is co-expressed with CA IX in neoplastic tissues such as mesotheliomas and choroid plexus tumors. Very high expression of CA XII has been demonstrated in low-grade ductal carcinoma and lobular carcinoma of the breast, and low-grade gliomas of the brain. CA XII expression is strong also in renal
clear-cell carcinomas, chromophobic cell carcinomas and oncocytic tumors (Ivanov et al., 2001; Parkkila et al., 2000). mRNA expression of CA XII has also been studied in cultured pancreatic tumor cell lines (Nishimori et al., 1999) and overexpression has been detected in the small-cell lung carcinoma cells. It has been reported also that most colorectal tumors seem to have abnormal CA XII expression in the deep parts of the adenomatous mucosa where the expression increased with the grade of dysplasia. The diffuse expression of CA XII in most malignant tumors correlates with their biological behavour (Kivelä et al., 2000). The expression of CA XII in gastric tumors is only slightly higher than in normal gastric mucosa (Leppilampi et al., 2003).
The carbonic anhydrase XII could serve as a biomarker for some malignant tumors (e.g.
non-small cell lung carcinoma) and could be considered a potential target for novel therapeutic applications (Ivanov et al., 1998). CA XII has been demonstrated to be a good prognostic marker in invasive breast carcinoma patients (Watson et al, 2003).
2.3.4. Von Hippel-Lindau
Both CA IX and XII are functionally related to von Hippel-Lindau-mediated carcinogenesis and down-regulated by expression of the wild-type von Hippel-Lindau tumor suppressor protein (pVHL). Germline mutations in the VHL genes of humans cause a hereditary cancer syndrome, which is called the von Hippel-Lindau disease (Kondo & Kaelin, 2001).
The solid tumours develop in two stages. In the first stage the malignant cells grow into small tumours, and as soon as they face hypoxia the growth is ended. In the second stage, hypoxia causes severe changes in gene expression and leads to clonal selection within the tumour cell population, which is followed by angiogenesis and fundamental changes in energy metabolism (respiration is replaced by glycolysis) (Dang & Semenza, 1999;
Hanahan & Folkman, 1996; Semenza, 2000; Stubbs et al., 2000; Warburg, 1930;).
Consequently, the tumour microenvironment shows low oxygen tension, high hydrostatic pressure, and acidic extracellular pH (Helmlinger et al., 1997; Jain, 1999). Expression of
genes for angiogenesis, energy metabolism and transmembrane CAs is controlled by the hypoxia-inducible transcription factor, HIF-1, which integrates pathways regulating physiological responses to acute and chronic hypoxia (Bunn & Poyton, 1996; Gleadle &
Ratcliffe, 1998; Gnarra et al., 1996; Hanahan & Folkman, 1996; Ivanov et al., 1998;
Kaelin & Maher, 1998; Maxwell et al., 1999; Ohh & Kaelin, 1999; Semenza, 1999;
2000). The ubiquitinproteasome proteolysis system releases cellular proteins from a multiprotein ubiquitin (Ub) ligase complex, VBC (VHL/Elongin B/ElonginC). pVHL is an integral part of the complex, and targets for the proteolytic degradation are HIF-1α and
HIF-2α which both bind pVHL and degradate in normoxic conditions, not in hypoxia Figure 2.6. The hypoxia-inducible factor pathway of hypoxia control. As tumor masses expand, they outrun their oxygen supply requiring the synthesis of new capillaries, more red blood cells, and a switch to anaerobic glycolysis in order to survive and progress through what would otherwise be a lethal threat.
Hypoxia-inducible factor (HIF-1α) is an unstable protein that fails to accumulate in cells except when they are exposed to a hypoxic environment.
Through inhibitory signals that are processed by the von Hippel-Lindau (VHL) protein, HIF-1α is stabilized, accumulates, and interacts with ARNT (aryl hydrocarbon receptor nuclear translocator), its physiological partner, to form an active transcription factor.
HIF-1α activates a number of cellular genes, including those for proteins that carry out anaerobic glycolysis, for erythropoietin (red blood cell production), and for vascular
endothelial cell growth factor (VEGF) (Modified from Livingston &
Shiydasani, 2001).
1999). Therefore pVHL is the cause of the hypoxia-driven changes in gene expression in tumors. In VHL patients CA12 gene is overexpressed in tumors because of the absence of pVHL (Ivanov et al., 1998). Ivanov et al. have shown in 2001 that hypoxia causes upregulation of CA12 expression in tumor cell line expressiong a normal VHL message (figure 2.6.).
2.4. ACATALYTIC CARBONIC ANHYDRASES
The carbonic anhydrase related proteins, CA-RPs, are inactive isoforms in which one or more of the three critical zinc ion binding histidine residues in the active site have amino acid substitutions. Even though the function of these acatalytic isoforms is unknown, high sequence homologies between human and mouse cDNAs suggest that they have biologically important roles in higher animals. Acatalytic CA domain forms a ligand- binding domain for the two members of the receptor-type protein tyrosine phosphatase (RPTP) family (RPTP-β and RPTP-γ). They might also have an association with cancer since one of the RPTPs (RPTP-γ) could function as a tumor suppressor protein (Barnea et al., 1993; Nishimori, 2004; Wary et al., 1993).
The mammalian α-CA gene family includes at least three CA-RPs. CA-RP VIII has been identified in the Purkinje cells of the cerebellum (Kato, 1990), and it has the most highly conserved amino acid sequence of the α-CAs between the human and mouse homologues (~98%) (Skaggs et al., 1993). The amino acid sequence of CA-RP VIII is deduced with changes in the catalytic site residues which could explain the loss of the CA activity of this isoform. The CA-RP VIII is highly expressed in the murine brain (the granular and molecular layers of the cerebellum, the pial surface and in the large neurons throughout the cortical layer, the medial habenulae, neurons of the thalamus, strongly expressed in the hippocampal formation, specifically in the pyramidal cells of the Ammon’s horn and in the granular cells of the dentate gyrus, and in the choroids plexus and cerebrospinal fluid-containing channels), liver, lung, heart, gut, and thymus (Lakkis et al., 1997; Ling et al., 1994, Skaggs et al., 1993). The CA-RP VIII is expressed also in the human testis, salivary glands, placenta and lungs (Ling et al., 1994; Skaggs et al., 1993). The
expression of murine CA-RP VIII mRNA is relatively high in the adult and fetal lung whereas it has not been found in the human adult lung tissue (Lakkis et al., 1997;
Akisawa et al., 2003). The CA-RP VIII is strongly expressed in non-small cell lung carcinomas (at the invasion front). This suggests that the CA-RP VIII would be an oncofetal antigen and would have a role in non-small cell lung carcinomas. The wide distribution of its mRNA in the mouse brain could suggest a non-specific but generalized function for CA-RP VIII in the brain tissue.
2.5. CARBONIC ANHYDRASES IN NERVOUS SYSTEM
Over sixty years ago Ashby (1943) demonstrated CA activity in mammalian brain where it has various physiological functions such as fluid and ion compartmentation (Bourke &
Kimelberg, 1975), the formation of cerebrospinal fluid (CSF) (Maren, 1967), seizure activity (Anderson et al., 1984), the respiratory response to carbon dioxide (Ridderstråle
& Hanson, 1985) and the generation of bicarbonate for biosynthetic reactions (Tansey et al., 1988; Cammer, 1991). Giacobini (1961; 1962) reported the first suggestions about glia being the main site of CA expression in the central nervous system (CNS). His experimentations on CA activity in glial cells of rat brainstem were made by dissection of neurons and glial cell clumps. In 1964 Korhonen et al. showed that the areas rich in myelinated fibres and glial cells of the mouse brain have the highest CA activity. When the glial cells and neurons were separated by bulk isolation, the higher CA activity was detected in the glia than in neurons (Sinha & Rose, 1971; Nagata et al., 1974). Significant CA activity was also observed in the myelin isolated from rat, mouse, monkey, cat, and rabbit brains (Cammer et al., 1976; 1977; Sapirstein & Lees, 1978). In bulk-isolated cells, the oligodendrocytes seemed to have the highest activities whereas the neurons and astrocytes had only very low activities (Snyder et al. 1983).
Immunostaining of rodent and human brains and spinal cords with antisera against CA II showed positive signal in the oligodendrocytes (Roussel et al., 1979; Ghandour et al., 1979; 1980; Langley et al., 1980; Kumpulainen & Korhonen, 1982; Kumpulainen et al., 1983) and in myelin sheaths (Roussel et al., 1979; Kumpulainen & Korhonen, 1982).
Low levels of CA has been observed in the astrocytes in cultured cells (Kimelberg et al., 1982), in bulk isolated cells (Snyder et al., 1983), and in one immunohistochemical study on brain tissue sections (Roussel et al., 1979).
In other studies variable amounts of CA II have been observed also in the astrocytes (in the normal grey matter of the brain), in reactive astrocytes (in severely gliotic white matter of the jimpy mutant mouse and of rats with experimental autoimmune encephalomyelitis), in jimpy oligodendrocytes, and in reactive astrocytes, oligodendrocytes and neurons surrounding brain tumours and other types of neoplastic cells of these tumours (Borelli et al., 1982; Nakagawa et al., 1986; 1987; Cammer &
Tansey, 1988; Ghandour & Skoff, 1988; Cammer, 1991; Jeffrey et al., 1991; Cammer &
Zhang, 1992). An observation of Nogradi (1993) showed that the active brain macrophages express CA II and III, and the resting microglial cells express only CA III.
The transformation of these microglial cells from a metabolically and/or immunologically more active form to less active (or vice versa), has been related to the immunoreactivity of CA II (Perry & Gordon, 1988; Ashwell, 1991; Nogradi, 1993).
CA activity has an important role in the production of CSF and in its regulation of pH and ionic constituents (Maren, 1967; Maren & Broder, 1970). The CA II and III activity has been observed in the cytoplasm and microvilli of the epithelial cells of the choroid plexus (Kumpulainen & Korhonen, 1982; Nogradi et al., 1993). The CA II activity and fatty acid synthase and acetyl-Coa carboxylase (first lipid synthesis enzymes) all exist in oligodendrocytes, which could suggest that CA II provides bicarbonate for the synthesis of fatty acids which form lipids to myelin (Tansey et al., 1988; Cammer, 1991). After the neurotoxic demyelination the CA activity is required for the compaction of myelin (Yanagisawa et al., 1990). The intracellular CA can be related to specialized sensory functions as it has been found in retinal neurons, in the sensory neurons of the ganglia in the peripheral nervous system, and in few CNS neurons (Neubauer, 1991). The localization of CA IV in the brain of adult rats and CA II-deficient mice is limited to the luminal surface of the capillary endothelial cells which could suggest an important role at the blood-brain barrier (Ghandour et al., 1992).
Table 2.2. Expression of CA isozymes in the brain.
Isozyme
Site of
expression
II oligodendrocytes
astrocytes
myelin
choroid plexus
neurons
III choroid plexus
microglial cells
IV endothelial cells
V astrocytes
neurons
VII choroid plexus
pia
neurons
thalamus
hippocampus
cerebellum
XIV neurons
Lakkis et al. (1997) demonstrated that there is CA VII mRNA expression in the pia, choroid plexus and neurons of the cortical layer, thalamus, and medial habenulae. A high expression can be seen in the pyramidal and granular cells of the hippocampus and in the cerebellum. Car7 and Car8 are transcribed to different degrees in the Purkinje cells;
lower expression has been observed in the molecular and granular cell layers.
Transcription signals of Car7 and Car8 are excluded from white matter regions.
The mitochondrial isoforms (CA VB) has been observed in astrocytes and neurons (Ghandour et al., 2000). This isozyme could have an important role in astrocytes in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase expressed in those cells (Yu et al., 1983). Two novel roles for neuronal CA V have been suggested such as the regulation of the intramitochondrial calcium levels and the regulation of neuronal transmission by facilitating the bicarbonate ion-induced GABA responses (Ghandour et al., 2000). The finding of evidence that carboxylation of pyruvate to malate occurs in neurons and that it supports formation of transmitter glutamate (Hassel, 2001)
has suggested that the previous predictions were not the final word in the neuronal metabolism of pyruvate, and that CA V might serve a number of different physiological processes in neurons.
Parkkila et al. (2001) demonstrated that CA XIV, a membrane-bound isozyme, is highly expressed in some neurons, highest expressed on large neuronal bodies and axons in the anterolateral part of the pons and medulla oblongata, in the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus..
The localization of CA XIV in neurons may have an important role in the production of alkaline shift linked to the neuronal signal transduction.
2.5.1. Formation of cerebro-spinal fluid (CSF)
The cerebro-spinal fluid (CSF) provides a protective buoyancy for the brain which effectively makes the weigth of the brain 1/30th of its actual weigth. The CSF also provides maintenance of the chemical environment of the central nervous system. The way in which the metabolites are removed is a one-way flow of CSF from ventricular system, around the spinal cord, into the subarachnoid space and into the venous sinuses.
Nowhere in the body there’s more need for homeostasis than in the brain. The mechanism for maintenance of this barrier function lies in the capillary network supplying blood to the brain. The blood-brain barrier (BBB) protects the brain against surging fluctuations in ion concentrations of the plasma (Davson et al., 1987).
The concept of blood-brain barrier shows that the BBB is located in endothelial cells of capillaries of the brain. These endothelial cells in the brain are different to those in peripheral tissues. Brain endothelial cells are joined by tight junctions of high electrical resistance. In brain endothelial cells there’s no movement like in peripheral endothelial cells. Brain capillaries are in contact with foot processes of astrocytes which separate the capillaries from the neurons. The BBB is both a physical barrier and a system of cellular transport mechanisms. It maintains homeostasis by restricting the entrances of potentially harmful chemicals from the blood, and by allowing the entrance of essential nutrients
(Davson et al., 1987). Lipid soluble molecules (such as ethanol) are able to penetrate trough the BBB relatively easily via the lipid membranes of the cells. Water soluble molecules (such as sodium) are unable to transverse the barrier without the use of specialized carrier-mediated transport mechanism (figure 2.9.). There are some areas of the brain that do not have a blood-brain barrier. The BBB is absent in the pituitary because it allows products to pass into the circulation. Another are is a chemoreceptive area which allowa transcellular transport for the water balance and other homeostatic functions (Laterra et al., 1991; Schmidley & Maas, 1990).
The ependymal cells that line the ventricles of the brain fuse with the pia mater to form the choroid plexus (figure 2.7.). The choroid plexusas are branched structures made up of numerous villi. They provide a large surface area for the secretion of CSF. The choroid plexus has a rich blood supply. Capillaries in the choroid plexus are highly specialized for their function. They are fenestrated and provide little resistance to the movement of small molecules. The epithelial cells of the choroid plexus are linked by junctional complexes, thus the epithelium forms the blood-CSF barrier (Johanson et al., 1995).
CSF is secreted by the epithelial cells of the choroid plexuses which are polarised so that the properties of their apical membrane (facing ventricle) differ from those of the basolateral membrane (facing blood). Both of these membranes have a greatly expanded area, so that the total area available for transport is similar to that of the BBB. CSF secretion involves the transport of ions (Na+, Cl- and HCO -) across the epithelium from
Figure 2.7.The picture on the left is a sagital section of a human brain showing the location of the choroid plexus in the ventricular system (Modified from http://cal.vet.upenn.edu/ne uro/server/slides/ns_075- Ch.jpg).
blood to CSF (figure 2.8.). This movement of ions creates an osmotic gradient that drives the unidireccional transport of ions across an epithelium. Secretion can occur because of the polarised distribution of specific ion transporters in the apical or basolateral membrane of the epithelial cells (Siegel et al., 1999).
The choroid plexus consist of highly vascularized masses of pia mater tissue that dip into pockets formed by ependymal cells (figure 2.9.). The preponderance of choroid plexus is distributed throughout the fourth ventricle near the base of the brain and in the lateral ventricles inside the right and left cerebral hemispheres. The cells of the choroidal epithelium are modified and have epithelial characteristics. These ependymal cells have microvilli on the CSF side, basolateral interdigitations, and abundant mitochondria. The ependymal cells, which line the ventricles, form a continuous sheet around the choroid plexus. While the capillaries of the choroid plexus are fenestrated, non-continuous and have gaps between the capillary endothelial cells allowing the free-movement of small molecules, the adjacent choroidal epithelial cells form tight junctions preventing most macromolecules from effectively passing into the CSF from the blood (Brightman, 1968;
Redzic & Segal, 2004).
Figure 2.8. Model of ion transport at the choroid plexus epithelium. Net transport of Na+ and Cl across the epithelium results in the secretion of CSF. Cl efflux from the epithelium to CSF is mediated by a cotransporter. It is uncertain whether that transporter is of the Na+/K+/2Cl or K+/Cl form. The generation of H+ and HCO 3 by carbonic anhydrase is important in the secretion of CSF (Modified from Siegel et al., 1999).
Figure 2.9. Blood CSF barrier. The blood CSF barrier is at the choroid plexus epithelial cells, which are joined together by tight junctions.The capillaries in the choroid plexus differ from those of the brain in that there is free movement of molecules across the endothelial cell through fenestrations and intercellular gaps. Microvilli are present on the CSF-facing surface and they increase the surface area of the apical membrane (Modified from Siegel et al., 1999).
2.6. CARBONIC ANHYDRASE INHIBITORS
Regulation of the acid-base balance is a physiological process, which involves a number of proteins such as ion transport proteins, plasma membrane receptors and their ligands, and CAs. Different CA isozymes have an important role in ion and water transport and some isozymes may physically interact with various ion transporters (Casey et al., 2004).
Carbonic anhydrase inhibitors (CAIs) can be classified into two groups: the metal- complexing anions and the unsubstituted sulfonamides. Sulfonamides are the most important CAIs because they bind in a tetrahedral geometry of the zinc ion and forms a network of hydrogen bonds involving many amino acids as well as the metal ion (Supuran, 2004). The major applications of CA inhibitors are used in opthalmology.
Acetazolamide, methazolamide, ethoxzolamide and dichlorophenamide are systemic antiglaucoma drugs, which inhibit CA II and CA IV present in the ciliary processes of eye. The inhibition of CAs prevents the sympthoms of glaucoma by reducing the
secretion of aqueous humor and HCO3- and lowering the intraocular pressure (Mincione et al., 2004).
Acetazolamide has been observed to inhibit both Na+ and Cl- absorption in human intestines (Turnberg et al., 1970a; 1970b), and this proposes that most of the absorption must be mediated by electroneutral Na+-H+ and Cl--HCO3- exchange processes. Because water absorption follows ion movements, are CAs probably also implicated in water absorption. Abundantly expressed in the non-goblet epithelial cells of the mammalian colon, CA I and II are probably key players in this physiological process (Lönnerholm et al., 1985; Parkkila et al., 1994). The luminal content of the colon is alkalized by bicarbonate secretion, which depends on apical Cl--HCO3- exchange (Feldman &
Stephenson, 1990) and it acidifies the luminal content by active proton secretion (Suzuki
& Kaneko, 1987). This proton secretion can facilitate non-ionic fatty acid uptake by promoting apical Na+-H+ exchange (Sellin & DeSoignie, 1990) or a proton ATPase pump (Gustin & Goodman, 1981).
There may be several CA isozymes involved in the regulation of the acid-base balance in the alimentary tract. The clinical applications of CA inhibitors have been limited in the gastrointestinal tract so far. One of the most attractive applications is that CA inhibitors could be useful for the therapy of peptic ulcer. An early approach to attack the machinery of the acid-producing cell by acetazolamide was discovered by Baron (2000). Davenport suggested in 1939 that CA might be essential for acid production, so an inhibitor of this enzyme would inhibit gastric acid secretion. A brief acid inhibition by acetazolamide was demonstrated and concluded that its action was too brief to be therapeutically useful (Janowitz et al., 1952; 1957). But later studies showed that acetazolamide might be effective in the treatment of gastric ulcer (Puscas et al., 1989; Erdei et al., 1990).
Acetazolamide has never generally been approved for the treatment of gastric ulcer because it has many unfavourable side effects and documentation of its efficacy has been insufficient.
The CA activity in renal acidification has been studied using CA inhibitors, although they have not been very useful in the therapy of renal diseases (Supuran & Scozzafava, 2000).
Acetazolamide, methazolamide, ethoxzolamide and dichlorophenamide can be used for the treatment of edema induced by drugs or congestive heart failure (Supuran &
Scozzafava, 2000) but they can cause numerous undesired side effects, such as metabolic acidosis, nephrolithiasis, CNS symptoms and allergic reactions (Tawil et al., 1993;
Supuran et al., 2001). The acute response to CA inhibition is an increase in the excretion of bicarbonate, sodium and potassium, an increase in urinary flow, and titratable acid (Bagnis et al., 2001) and the loss of bicarbonate and sodium is considered self-limited on continued administration of the inhibitor, probably because the initial acidosis resulting from bicarbonate loss activates bicarbonate reabsorption via CA-independent mechanisms. Chronic CA inhibition stimulates morphologic changes in the collecting ducts (Bagnis et al., 2001) and therefore, CA activity could play an important role in determining the differentiated phenotype of renal epithelium.
2.6.1. CA inhibitors in the nervous system
Figure 2.10. Acetazolamide.
CA inhibitors have profound effects on the function of the central nervous system (CNS).
Acetazolamide (figure 2.10.) can reduce CSF (cerebro-spinal-fluid) production by about 50 per cent (Maren, 1972; McCarthy & Reed, 1974), the concentration of carbon dioxide in brain tissues increases and probability of seizures decreases. It is also known that it dilates intracranial vessels (Maren, 1967; Hauge et al., 1983) which increases cerebral blood volume (CBV). This increase in CBV reflects an intrinsic volume load to the intracranial cavity and with normal CSF circulation and absorption it does not elevate significantly the intracranial pressure (Parkkila et al., 2004).
